Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. See, e.g., PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; and 5,712,146; and Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by PKS enzymes. These enyzmes, which are complexes of multiple large proteins, are similar to, but distinct from, the synthases which catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by "PKS gene clusters." PKS gene clusters usually consist of three or more open reading frames ("ORFs"), each comprising two or more "modules" of ketosynthase activity, each module of which consists of at least two (if a starter unit) and more typically three or more enzymatic activities or "domains." Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis of the polyketide synthesized. These two major types of PKS enzymes are commonly referred to as Type I or "modular" and Type II "aromatic" PKS enzymes.
The FK-520 PKS enzyme is a member of the Type I or modular PKS enzyme group. In this type, a set of separate catalytic active sites (each active site is termed a "domain", and a set thereof is termed a "module") exists for each cycle of carbon chain elongation and modification in the polyketide synthesis pathway. The active sites and modules of a typical Type I PKS enzyme are shown in FIG. 9 of PCT patent publication No. WO 95/08548, which depicts a model of 6-deoxyerythronolide B synthase ("DEBS"), which is involved in the synthesis of erythromycin. Six separate modules, each catalyzing a round of condensation and modification of a 2-carbon unit, are present in DEBS. The number and type of catalytic domains that are present in each module varies, and the total of 6 modules is provided on 3 separate proteins (designated DEBS-1, DEBS-2, and DEBS-3, with 2 modules per protein). Each of the DEBS polypeptides is encoded by a separate open reading frame (ORF). See Caffrey et al., 1992, FEBS Letters 304: 205, incorporated herein by reference. The catalytic domains of the DEBS polypeptides provide a representative example of Type I PKS structure. In this particular case, modules 1 and 2 reside on DEBS-1, modules 3 and 4 on DEBS-2, and modules 5 and 6 on DEBS-3; module 1 is the first module to act on the growing polyketide backbone, and module 6 the last.
A typical (non-starter) minimal Type I PKS module is typified by module 3 of DEBS, which contains a ketosynthase ("KS") domain, an acyltransferase ("AT") domain, and an acyl carrier protein ("ACP") domain. These three enzyme activities are sufficient to activate the 2-carbon extender unit and attach it to the growing polyketide molecule. Additional domains that may be included in a module relate to reactions other than the actual condensation, and include a ketoreductase activity ("KR") activity, a dehydratase activity ("DH"), and an enoylreductase activity ("ER"). With respect to DEBS-1, the first module thereof also contains repeats of the AT and ACP activities because it catalyzes initial condensation, i.e. it begins with a "loading domain" represented by AT and ACP, which determine the nature of the starter unit.
The "finishing" of the 6-deoxyerythronolide molecule is regulated by a thioesterase ("TE") activity in module 6. The TE activity catalyzes cyclization of the macrolide ring by formation of an ester linkage. In FK-506, FK-520, rapamycin, and similar polyketides, the ester linkage formed by the TE activity is replaced by a linkage formed by incorporation of a picolate acid residue. The enzymatic activity that catalyzes this incorporation for the rapamycin enzyme is known as rapP.
In PKS polypeptides, the regions that encode enzymatic activities (domains) are separated by linker or "scaffold"-encoding regions. These scaffold regions encode amino acid sequences that space the enzymatic activities (domains) at the appropriate distances and in the correct order. Thus, the linker regions of a PKS protein collectively can be considered to encode a scaffold into which the various domains (and thus modules) are placed in a particular order and spatial arrangement. Generally, this organization permits PKS domains of different or identical substrate specificities to be substituted (usually at the DNA level) between PKS enzymes by various available methodologies. Thus, there is considerable flexibility in the design of new PKS enzymes with the result that known polyketides can be produced more effectively, and novel polyketides useful as pharmaceuticals or for other purposes can be made.
Additional structural complexity in the resultant polyketides arises from or can be introduced by various activities, including glycosylation, hydroxylation, methylation, and other enzymatic activities. The rapP enzymatic activity mentioned above is an example of one such activity; another example is the hydroxylation of a polyketide by an oxidase enzyme similar in structure and function to the cytochrome P450 oxidase enzyme. By appropriate application of recombinant DNA technology, a wide variety of polyketides can be prepared in a variety of different host cells provided one has access to nucleic acid compounds that encode PKS proteins and polyketide modification enzymes. The present invention helps meet the need for such nucleic acid compounds by providing recombinant vectors that encode a PKS enzyme and various polyetide modification enzymes from an FK-520 producing strain of Streptomyces hygroscopicus.